In the interest of improving cancer treatment, considerable attention has been placed on the modification of radiation damage. The interaction of a variety of chemotherapy and/or molecularly targeted agents with radiation is under study to determine if tumors can be made more sensitive or normal tissues more resistant to radiation treatment. The central aim is to identify approaches that will result in a net therapeutic gain, thus improving cancer treatment with radiation. One goal of the project is to define and better understand those aspects of tumor physiology, including cellular and molecular processes and the influence of the tumor microenvironment on treatment response. The ability to enhance the response of the tumor to radiation, without enhancing normal tissue within a given treatment field is desirable. We finished pre-clinical studies of a novel HSP90 inhibitor and a CDK4/6 inhibitor as a radiation sensitizers (in vitro and in vivo). Significant in vitro radiation dose modification factors have been observed with both agents and xenograft studies indicate that the combination of the drug with fractionated radiation results in enhanced radiosensitivity. Further we have identified molecular biomarkers that will be helpful in assessing treatment efficacy for both agents. Both agents have potential of being translated into human clinical trials. We continue to evaluate a number of metabolic inhibitors as radiation modifiers under the working hypothesis that inhibition of metabolism (for example, decreased ATP production) will diminish the repair of radiation-induced DNA damage. In vitro studies have shown that a novel lactate dehydrogenase inhibitor (LDHAi) enhances the radiosensitivity of human pancreatic carcinoma cells. Preliminary xenograft studies with this agent have shown a significant tumor growth delay with drug alone, but no enhancement of the radiation response. We are currently exploring different drug delivery routes to determine if this might provide enhancement of radiosensitivity. We have initiated studies combining the LDHAi with an inhibitor of pyruvate dehydrogenase. Preliminary studies shown significant enhancement of radiosensitivity in vitro. Xenograft studies combining both agents with radiation are planned as well as functional xenograft metabolic imaging studies using 13C-pyruvate to determine if particular biochemical pathways are blocked. These pre-clinical studies will provide the necessary information to consider these agents in a clinical human trial for tumor radiosensitization. Studies conducted last year demonstrating that rapamycin supplied in the animal's chow protected against radiation-induced lung fibrosis in part by preventing radiation-induced stem cell senescence. This finding has prompted us to study radiation-induced senescence in human tumor cells. High doses of radiation result in a large percentage of cells becoming senescent. With the recent development of senolytic agents, which can kill senescent human cancer cells, we have conducted xenograft studies where tumors receive large doses of radiation and are subsequently treated after radiation with senolytic agents. We have found that a new class of molecularly targeted agents, namely, CDK4/6 inhibitors are potent radiation sensitizers both in vitro and in vivo. Interestingly, these agents also inhibit tumor blood vessel formation (vasculogenesis) post-radiation treatment further increasing treatment related tumor growth delay. A major emphasis will be placed on determining the mechanism of vasculogenesis inhibition in future studies. Collectively, we have identified a number of pre-clinical approaches to initiate human radiation oncology clinical trials for modulation of radiation effects on tumors.